Much of biology is regulated by macromolecular interactions. Understanding the principles that govern the regulation of macromolecular interactions is critical for the rational manipulation of biological processes such as the immune response, gene expression and cellular growth. Allosteric proteins, which alter the interactions between subunits in response to environmental conditions, provide ideal systems for exploring the regulation of macromolecular interactions. We will be using three disparate invertebrate and primitive vertebrate hemoglobins as model systems for investigating atomic-level principles of allosteric protein function. Despite the similar tertiary structure of these hemoglobins, the extent of assembly into cooperative complexes is very different. The goal of this project is to elucidate the structural diversity and common themes that operate to regulate function in this family of proteins. Scapharca dimeric hemoglobin represents the simplest possible model system for cooperative protein function. Our powerful combination of structural, functional and mutational approaches for exploring the cooperative mechanism has revealed a number of important aspects of its function, including the central role of water molecules as sensors of ligation state. We are continuing our approaches to learn how assembly alters ligand pathways and to dissect the coupling between alternate pathways for communication between subunits. A much more complex system is Lumbricus erythrocruorin, which is assembled from 144 hemoglobin subunits and 36 non-hemoglobin (linker) subunits. Our structural results reveal that this molecule is assembled using an intricate hierarchy of symmetry. Our investigation of this molecule is designed to determine the role of two important domains, coiled coils and the cysteine-rich LDL-A module, for formation of this gigantic complex. In addition, we will determine the structural basis for the high cooperativity of this complex. A third system under investigation is the hemoglobin from lamprey, a primitive vertebrate. Unlike other hemoglobins, this hemoglobin gains cooperativity as a result of the concentration dependent equilibrium between different oligomeric states. Our structural analysis suggests a number of hypotheses for the regulation of function, which we will be testing by mutagenesis, kinetic and structural experiments.